Production, crystallization, and structure solution of 2909 Fab.
Transient transfection of plasmids encoding heavy and light chains of antibody 2909 generated ~80 mg of purified 2909 immunoglobulin per liter of cells. For the 2909 light chain produced by transient transfection, N-terminal sequencing showed cleavage to occur after the native N-terminal serine, likely a consequence of the signal peptide chosen. There was no difference, however, in neutralization activity by 2909 antibody produced from hybridomas or by transient transfection. Reduction, alkylation, and Lys-C proteolytic generation of the 2909 Fab, meanwhile, gave overall yields of 0.4 mg of Fab per mg of immunoglobulin. Crystals of 2909 Fab typically appeared a few days after setup and grew as hexagonal plates to maximum dimensions of 0.20 by 0.20 by 0.03 mm within 2 weeks.
Crystals were radiation sensitive, and diffraction intensities dropped off substantially after 5 Å. Nonetheless, a complete trigonal data set to 3.3 Å was collected with 0.5° oscillations by using a 0.05-mm pinhole and translating the crystal to a fresh unexposed position every ~25° of data collection.
Molecular replacement with the previously determined PG16 structure (PDB ID 3LRS) (31
) showed the space group to be P32
21, with unit cell dimensions of a
= 180.4 Å and c
= 222.5 Å, and to contain 6 Fab molecules in the asymmetric unit. Initial rigid body and TLS refinement in PHENIX (1
) yielded an R
factor of 40% (Rfree
= 42%) and produced electron density maps that clearly showed the missing CDR loop regions. Final refinement to 3.3 Å with PHENIX (1
) yielded an R
factor of 24% (Rfree
= 30%). Data collection and refinement statistics are shown in Table .
Data collection and refinement statistics
Overall structure of 2909 Fab.
The six copies of 2909 Fab in the asymmetric unit refined to nearly identical conformations. Pairwise superposition of the Fab molecules yielded root-mean-square-deviation (RMSD) values ranging between 0.5 and 1.1 Å for all atoms (see Fig. S1 in the supplemental material). Several residues at the CDR H3 loop apex (residues 100 to 100h) were disordered in two of six copies of the Fab, and residues 127 to 133 in the heavy chain constant region were disordered in all of the molecules in the asymmetric unit; these residues were excluded from the final model. The structural analysis presented here is based on Mol1 (chains H and L for heavy and light, respectively), which exhibited the most clearly defined electron density and lowest overall B factors. The final Mol1 model contained heavy chain residues 1 to 126 and 134 to 214 and light chain residues 2 to 208; notably, the entire CDR H3 of Mol1 was reasonably defined at the main-chain level.
2909 CDR H3 loop.
The structure of the 2909 Fab revealed an antigen-combining site dominated by a protruding CDR H3 subdomain that extends ~20 Å beyond the rest of the combining loops and comprises almost 40% of the combining region surface area (Fig. ). As defined by Kabat nomenclature and numbering (see Materials and Methods) (19
), CDR H3 of 2909 is 21 residues in length, roughly 50% longer than the medium length of human CDR H3s. When measured from tip to base, the CDR H3 loop is ~30 Å in length (Fig. ). Contiguous electron density was observed for much of the loop backbone, although many side chains, particularly at the apex of the loop, are not well defined (Fig. ). The CDR H3 loop is stabilized at its base with interactions from other heavy and light chain residues, while ~70% of the loop is solvent accessible (Fig. ).
FIG. 1. Overall structure of 2909 Fab. The 2909 crystal structure reveals a combining region dominated by a protruding, acidic CDR H3 loop. (A) Ribbon representation of the 2909 Fab structure is shown, with heavy and light chains colored in blue and green, respectively. (more ...)
FIG. 2. Details of 2909 CDR H3. 2909 CDR H3 is a 21-residue loop forming a β-hairpin structure. The loop is stabilized at the base in a bulged conformation by other CDR loop residues. Most of the CDR H3 loop extends out from the combining site and is (more ...)
Within the crystallographic asymmetric unit, the tip of the CDR H3 loop for any given Fab is found in close proximity to one or two other neighboring Fab molecules. Despite slightly different crystal packing environments, the structure of the CDR H3 loop is nearly identical in each of the four Fab molecules that exhibit an ordered loop, with pairwise RMSD values ranging from 0.3 to 0.8 Å for Cα atoms. The overall structure of the loop did not appear to be significantly influenced by any direct contacts between loop residues and neighboring molecules within the crystalline environment.
The base of the CDR H3 loop forms a bulged conformation stabilized by hydrogen bonding and stacking interactions with other CDR loop residues (Fig. ). AspH95 is at the center of the bulge and interacts with HisH35 from the CDR H1 region. TyrL50 extends from the CDR L2 to hydrogen bond with AspH98. ArgH56 from the CDR H2 loop represents one of the few somatic mutations present in the 2909 gene (see Fig. S2 in the supplemental material) and stabilizes the CDR H3 loop through π-cation interactions with the phenyl ring of TyrH100i and hydrogen bonding with TyrH100k. The central portion of the loop is maintained in an antiparallel β-sheet structure composed of the residues AspH98, SerH99, and AspH100 from the ascending strand which hydrogen bonds to SerH100h, TyrH100i, and PheH100j on the opposite strand (Fig. 2B). A five-residue turn forms the tip of the CDR H3 loop. The electron density was weak for residues at the loop apex, and high B factors suggested inherent flexibility for this region.
Structural homology of 2909 CDR H3.
In a search of the Protein Data Bank, 2,272 entries were found to contain structural homologs of 2909 CDR H3, suggesting that β-hairpins of this geometry and length are relatively common in protein structures. A notable feature of 2909 CDR H3 is that much of the loop is exposed (67.1%). Histogram analysis of the solvent accessibility of all structural homologs showed a broad distribution, with two-thirds of the structures falling into the lower half of the distribution (Fig. ). Only 22 loop structures have ≥60% of their surface exposed. Among these 22 structures, 20 formed complexes by using the loop region to pair with or to pack against β-sheets of other subunits in the complex. The two remaining monomeric structures, 1S7I and 1XJC, have unknown functions. We also examined the sequence similarity of structural homologs to 2909 CDR H3. No structural homolog had a sequence identity to 2909 CDR H3 of greater than 30%, with two-thirds of all homologs sharing less than 15% identity (Fig. ). Overall, the exposed nature of the long β-hairpin makes 2909 CDR H3 an unusual structural motif in the general population of protein structures.
FIG. 3. Structural homology of 2909 CDR H3 region. Sixty-seven percent of the long β-hairpin structure of the 2909 CDR H3 loop is exposed to solvent, making it a rare structural motif among all protein structures. Structural homologs to the CDR H3 show (more ...)
Additionally, CDR H3 of 2909 was compared to long CDR H3 regions found in other antibodies, using a database based on the SACS server (http://www.bioinf.org.uk/abs/sacs/
). The 2909 CDR H3 loop was structurally aligned to the CDR H3 of each antibody in the database (the closest homologs at least 16 residues in length are given in Table S1 in the supplemental material), with 12 out of the 23 antibodies listed being anti-HIV-1 antibodies. One of the closest structural homologs identified was CDR H3 of anti-V3 antibody 447-52D, which gave an average RMSD of 1.76 Å over Cα atoms and an alignment of 19 (out of 20) residues (Fig. ). The second-best match was the anti-gp41 antibody Z13e1, which showed an RMSD of 1.63 Å over Cα atoms for 17 aligned residues. Despite the structural similarity, 447-52D and Z13e1 use their CDR H3 loops to interact with their respective epitopes in different ways: 447-52D uses one of its CDR H3 strands to pair with the V3 loop primarily through backbone hydrogen bonds and a few side chain interactions, while the Z13e1 CDR H3 packs against the gp41 loop through side chain interactions. In light of the high degree of structural homology between 2909 and 447-52D and the fact that the 2909 epitope includes the V3 loop, it is possible that part of the 2909 CDR H3 loop interacts with V3 in a way similar to that of 447-52D (although the alternative mode of recognition used by Z13e1 indicates that other possibilities cannot be excluded).
Anionic character of CDR H3 loop and tyrosine sulfation.
The 2909 CDR H3 loop sequence is tyrosine rich within an overall acidic context (Fig. ). This combination of tyrosine residues and anionic character is similar to the sequence signature recognized by human sulfotransferases, which introduce O sulfation of tyrosine as a posttranslational modification (28
). The precise sequon for O sulfation by sulfotransferase is not known, but reasonable predictions have been established with neural-network-based software programs, such as Sulfinator (27
) and Sulfosite (5
). With 2909, both programs predict sites of tyrosine sulfation, at TyrH100a
, at the middle and tip of CDR H3, respectively. Although additional sites are predicted by Sulfinator, both TyrH100a
are solvent exposed, increasing the likelihood that they are O sulfated (see Table S2 in the supplemental material). Electron density was not well defined for these residues, especially for TyrH100a
, where the entire phenol ring appears to be conformationally disordered, thereby preventing crystallographic confirmation of O sulfation (Fig. ). Nonetheless, the presence of two sites of sulfotyrosine modification on the 2909 heavy chain was confirmed by electrospray ionization mass spectrometry (ESI-MS) analysis (Table and Fig. ).
FIG. 4. Electrospray ionization mass spectrometry (ESI-MS) data for 2909 Fab. ESI-MS intact mass measurements of 2909 Fab are consistent with two sites of tyrosine sulfation in the heavy chain. Deconvoluted ESI mass spectra are shown for 2909 Fab heavy and light (more ...)
We decided to test the functional role of TyrH100a and TyrH100c by mutating the tyrosine to phenylalanine at both sites and assaying the mutants for neutralization (Table ). Mutational substitution of Phe for TyrH100a in the middle of the CDR H3 ablated neutralization activity in all 5 isolates tested that were sensitive to 2909. Substitution of Phe for TyrH100c at the tip of the loop ablated neutralization activity against SF162 and YU2.N160K and diminished neutralization (20- to 25-fold increase in IC50) against three other 2909-sensitive pseudoviruses containing the N160K mutation (Table ). The results indicate a critical role for these tyrosines, both of which are located on the heavy chain-facing side of the CDR H3.
IC50 neutralization values for 2909 CDR H3 tyrosine mutants
Comparison of antibodies 2909 and PG16.
Unlike antibody 2909, antibody PG16 is broadly reactive and can neutralize 70 to 80% of HIV-1 isolates (40
). Nevertheless, 2909 and PG16 appear functionally related: mapping reveals recognition of a similar V2-V3 epitope, distinguished by a requirement for Lys160 in the V2 loop for 2909 and Asn160 for PG16. Antibodies 2909 and PG16 use different light chains (IGLV3-21*01 and IGLV2-14*01, respectively) but share 68% sequence identity in their variable heavy chain genes, both of which are derived from VH3 gene precursors (IGHV3-43*01 and IGHV3-33*05 for 2909 and PG16, respectively) and the IGHJ6*03 gene (Fig. ).
FIG. 5. Sequence and structural comparison of 2909 with PG16. 2909 and PG16 are both from VH3 gene families. 2909 and PG16 both have critical residues in similar positions in their CDR H3 sequences, and these residues are located at approximately the same distance (more ...)
To determine whether the sequence and functional similarities between 2909 and PG16 correlate with the use of common structural elements, we compared the 2909 structure to the recently determined crystal structure of PG16 Fab (31
). Structural comparison of the two antibodies revealed common features. Both 2909 and PG16 have long, acidic CDR H3 loops which form subdomains that protrude from the antibody surface (Fig. ) and comprise significant portions of the combining surface: in 2909, CDR H3 contributes 36% of the CDR surface area; in PG16, CDR H3 contributes 42%. Moreover, CDR H3s in both PG16 (33
) and 2909 are tyrosine sulfated.
PG16 accommodates an additional 7 amino acids at the loop tip, giving it an overall hammerhead or axe shape (31
), rather than the headless axe or club shape observed in the 2909 structure (Fig. ). In both cases structural homologs to the CDR H3 were identified, and in both cases homology analysis indicated that a high degree of solvent exposure was unusual.
Critical residues in the CDR H3 domain for 2909 and PG16 are found at approximately the same distance from the rest of the combining site surface in both antibodies (Fig. ). For PG16, the most critical residue identified by mutational analysis is AspH100i
; replacement of this residue by alanine greatly diminishes PG16 neutralization activity (33
). For 2909, the potentially tyrosine sulfated TyrH100a
is critical for activity. Thus, critical negative charges reside in highly similar places in the 2909 and PG16 paratopes (Fig. ).
A closer look at the electrostatics surrounding the antigen-combining site revealed an acidic patch, including CDR H3 for both PG16 and 2909 (Fig. ). Interestingly, the negatively charged surfaces were found on opposing faces of each antibody, with CDR L2 residues making additional anionic contributions in 2909 and CDR H2 residues contributing in PG16.
If 2909 and PG16 were superimposed based on electrostatic similarities, the heavy chain of 2909 would align with the light chain of PG16 and vice versa (Fig. , bottom panel). Notably, such a superposition would also orient the functionally critical AspH100i
in PG16 and the functionally critical TyrH100a
on similar faces of their respective CDR H3s. For PG16, the tyrosine-sulfated residue (TyrH100h
) is not so critical for PG16 function, with replacement by phenylalanine at this site in PG16 resulting in only a 2.2-fold increase in the IC50
). The location of negative charge at a particular place in the paratope may thus be more critical than the precise character of the side chain.
Chimeric analysis of antibodies 2909 and PG16.
In the absence of structural information for quaternary-specific antibodies complexed with their epitopes, we used data from antibody chimeras to gain insight into how the CDR H3, heavy chain, and light chain elements contribute to the 2909 paratope. In this manner, regions within 2909 could be probed individually within the context of a related quaternary-specific antibody. Differences in sequence could be mapped out on models of chimeric antibodies that complement functionally to delineate functional hotspots.
To assess the individual contributions of heavy and light chain elements to 2909 and PG16 activity, heavy and light chains were swapped and the chimeric antibodies were assayed for neutralization against 2909-sensitive and PG16-sensitive pseudoviruses. Both chimeric antibodies could be produced, as verified by SDS-PAGE analysis of heavy and light chains, although the 2909H/PG16L swap expressed at low levels, suggesting that structural differences between the 2909 and PG16 light chains may affect the dimer interface. Neither chimeric antibody was active against SF162 and N160K mutant pseudoviruses or any PG16-sensitive pseudovirus (Table ).
IC50 neutralization valuesa for 2909/PG16 chimeras
To test the role of CDR H3 in neutralization, we swapped the CDR H3 sequence between 2909 and PG16 and assayed the chimeras for activity. A chimera composed of 2909 with the PG16 CDR H3 loop (2909H_PG16CDRH3/2909L) was not active against any isolate tested (Table ). A PG16 variant containing 2909 CDR H3 (PG16H_2909CDRH3/PG16L) did not express. Threading analysis and structural modeling of the chimera suggested that residues from the PG16 light chain might clash with 2909 CDR H3, thereby affecting the dimer interface and CDR H3 conformation. To further stabilize the PG16H_2909CDRH3 variant, site-directed mutagenesis was used to generate two variants of the PG16 light chain (PG16L-R96V and PG16L-D50V/L91W/R96V) that contained mutations to optimize interactions between the heavy and light chains. The redesigned chimeras also did not express. When the CDR H3 loop from each antibody was paired with its native light chain, however, neutralizing activity was recovered, although at a considerably reduced level. For example, when the PG16 heavy chain containing the 2909 CDR H3 loop was paired alongside the 2909 light chain (PG16H_2909CDRH3/2909L), the chimera exhibited weakened activity against select 2909-sensitive pseudoviruses (Table , last column). Similarly, when the 2909 heavy chain containing the PG16 CDR H3 was paired with the PG16 light chain (2909H_PG16CDRH3/PG16L), PG16-like activity was recovered for a single isolate, albeit at ~1000-fold reduced potency (Table , next to last column). Although the data suggest a role for the light chain in antigen recognition, they do not indicate whether the light chain is contributing primarily a functional role or a structural role in maintaining CDR H3 loop conformation.
Relationship of antibody 2909 to quaternary-specific rhesus monoclonal antibodies.
2909-like rhesus antibodies have been isolated that are strain specific in their activity toward SF162 and recognize V2-V3 quaternary neutralizing epitopes, which are present only on HIV-1 virions and infected cells (36
). To further investigate the functional similarity to 2909, the rhesus antibodies were assayed for neutralization activity against two 2909-sensitive pseudoviruses. As expected, all rhesus antibodies were extremely potent against SF162 (Table ). While 2909 can also neutralize YU2 containing an N160K substitution (16
), none of the rhesus antibodies showed activity against the mutant YU2 pseudovirus (Table ).
IC50 neutralization values for 2909/rhesus antibody chimeras
Next, chimeric antibodies were used to gain insight into the degree of similarity between 2909 and the quaternary-specific rhesus antibodies. Heavy and light chain swaps generated between 2909 and the rhesus antibodies were tested against 2909-sensitive isolates. All of these chimeras were expressed and could be purified, thereby suggesting that any sequence differences between 2909 and rhesus genes did not prevent chain association or have a major effect on the structural integrity of the antibody. Chimeras containing a rhesus heavy chain paired with the 2909 light chain retained some level of neutralizing activity against SF162 but were not active against the YU2.N160K pseudovirus (Table ). For three of the six rhesus-H/2909-L chimeric antibodies, little loss of activity was noted, while the remaining three showed an IC50 increase of 2,000- to 17,000-fold. Notably, the 2.5B heavy chain could be combined with the 2909 light chain with virtually no loss in neutralization potency against SF162. The immunoglobulin gene usage and sequence homology to 2909 are variable among all members of the rhesus group (see Appendix S1 in the supplemental material) and did not correlate with the degrees of activity exhibited by the different chimeras.
Chimeric antibodies comprising the 2909 heavy chain and a rhesus light chain were not active against any of the isolates tested. Several of the rhesus light chain sequences share ~90% sequence similarity to the 2909 light chain. To gain insight into the lack of function for chimeras composed of 2909 heavy chain and rhesus light chains, all of the chimeras were threaded onto the 2909 structure (see Table S3 in the supplemental material). Threading analysis suggested a potential explanation for the lack of complementation related to the heavy-light chain interface: residue 96 of the 2909 light chain is a valine, which can accommodate large side chains from the 2909 heavy chain. In contrast, rhesus antibodies have larger amino acids at this position in their light chain, which may be incompatible with the 2909 heavy chain.